Anti mouse anti goat igg igm isotype control

Idua^−/− miPSC lines, L13, L14, and L15, and wild-type control miPSC lines were trypsinized into a single cell suspension and plated onto a low attachment 6-well culture plate with basal mES media (without LIF) for 3 days to induce embryoid body (EB) formation. Further spontaneous differentiation was induced by culturing EBs by supplementing retinoic acid (100 μM) or 0.1% dimethyl sulfoxide. Each culture condition was reproduced in the 4-well chamber slides for immunofluorescent analyses. Antibodies for b3-tubulin (MA1-118, Invitrogen), α-actinin (AT6/172, abcam), α-fetoprotein (F1-6P2A8-P2B9A9, Invitrogen), and anti-mouse/anti-goat IgG/IgM isotype control (BD Pharmigen) were used as primary antibodies.

Pluripotent stem cell characteristics of the three Idua^−/− miPSCs were validated with immunofluorescent staining for stem cell marker proteins. Cells were plated on 8-well chamber slides coated with Matrigel until colonies formed. Slides were then fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton-X, and incubated with 5% Goat Serum. Antibodies for SSEA-1 (MC480 mAB, Thermo Fisher Scientific), Oct4a (C30A3 rabbit mAB, Cell Signaling Technology), and anti-mouse/anti-goat IgG/IgM isotype control (BD Pharmigen) were used as primary antibodies. Alexa Fluor 594 goat-anti-mouse IgG and Alexa Fluor 488 goat-anti-rabbit IgG were used accordingly as secondary antibodies. ProLong® Gold Antifade Reagent with DAPI (Life Technologies) was used for nuclear counter staining and mounting with coverslips.